Project Abstract Vascular mechanotransduction has long been recognized as mediating physiological processes such as flow-dependent dilation and pathological processes such as the localization of lesions in atherosclerosis. There is a consensus that hemodynamic forces represent two distinct biomechanical stimuli; that is, while unidirectional laminar fluid shear is atheroprotective, rapidly changing and/or reverse fluid shear stress (coupled with low average shear) is atherogenic. The overall goal of this application is to determine the molecular basis of mechanochemical signal transduction and the endothelium's ability to discriminate between these two biomechanical stimuli. We have already demonstrated that the atherogenic/inflammatory hemodynamic signaling originates from a macromolecular complex assembled around PECAM-1 at the endothelial cell-cell junction. One of the objectives of this application is to define the organization of the macromolecular complex located at the endothelial cell-cell junction and elucidate its mechanism of activation. The second objective is to investigate the hypothesis that the structural geometry of the junction regulates the sensitivity of the macromolecular complex to rapidly changing shear and confers the ability to discriminate between unidirectional and oscillatory/reverse flow. This project will use an entirely integrative approach using molecular biology, cell biology and biochemistry, vital cell imaging, and cell biomechanics. In particular, the specific aims are: 1) To investigate the interaction of PECAM-1 with VE-cadherin and VEGFR2 at the molecular level and the regulation of mechanochemical signaling through this interaction; 2) Determine the role of junctional GPCRs and G-proteins in mechanochemical signaling and their regulation by PECAM-1; 3) Characterize the geometry of the cell-cell junction by measuring its angle of inclination in in vivo and flow-adapted in vitro endothelium; and 4) Determine whether junctional inclination regulates endothelial response to reverse and oscillatory flow. The understanding of the molecular and structural mechanisms by which the endothelium senses oscillatory and reverse flow is crucial for the development of targeted therapies for flow-induced vascular inflammation and atherogenesis.